Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

Abstract

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454-1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204-211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration.

Membrane lipid domains and membrane lipid domain preparations: are they the same thing?

“…The observation that glycosphingolipids form clusters at the cell surface, which have been visualized by immuno-electron microscopy, represented one of the roots leading to development of the lipid raft hypothesis (4). Glycosphingolipid clustering in cell membranes was shown for globoside in human erythrocytes (5), polysialogangliosides in sh brain neurons (6), GM3 ganglioside in peripheral human lymphocytes and Molt-4 lymphoid cells (7) and LacCer in human neutrophils and neutrophilic diŠerentiated HL-60 cells (8). This method allows to deˆne the topology of a putative lipid raft marker respect to cell architectural features, and the application of image quantitative analysis allows to obtain information about the average lipid raft size and the total area of the membrane occupied by lipid rafts, however it requires an extensive sample manipulation, raising concerns about possible artefacts, and is not suitable for dynamic studies in time and space.…”

“…Anti-GD3 ganglioside monoclonal antibody R24 was used to isolate a DRM fraction from diŠerentiated rat cerebellar neurons (74). AntiLacCer monoclonal antibody Huly-m13 was used to isolate LacCer-enriched domains from human neutrophils, demonstrating the functional coupling between LacCer and Lyn (one of the best example of a detergent-resistant interaction conˆrmed by immunoelectron microscopy studies) (8). Two membrane subfractions were separated from Triton X-100-resistant membrane fractions from B16 melanoma cells by anti-GM3 ganglioside monoclonal antibody DH2 and by anti-caveolin antibody, respectively (143).…”

Role of lactosylceramide-enriched membrane microdomains in CD11b/CD18-mediated neutrophil phagocytosis

“…As observed in Fig. 2A, the Src (6,8). Therefore, the association between LacCers and Lyn molecules via its C24 fatty acid chains is considered to be indispensable for LacCer-enriched membrane microdomain-mediated neutrophil functions.…”

Special issue of “Organization and functions of lipid membrane microdomains”

“…Probably the most studied membrane microdomains are``lipid rafts'', membrane lipid domains dened by their sphingolipid-and cholesterol-rich nature, enrichment in glycosylphosphatidylinositol (GPI)-anchored proteins and membrane-anchored signaling molecules, and cytoskeletal association (3). The ability of glycosphingolipids to form clusters, suggested by many studies on artiˆcial membrane models (4), has been conrmed in intact cells by immunoelectron microscopic analysis use of the ultrathin-section method without organic solvents or SDS-treated freeze-fracture replica method (5,6). Glycosphingolipid segregation is probably one of the main driving forces leading to the formation of lipid membrane microdomains.…”

“…The ceramide structure of each glycosphingolipid is highly variable (7), whereas GPI-anchored proteins contain C18 and/or C16 fatty acid chains. In particular, the C18:0 fatty acid chain at the sn-2 position of GPIanchored proteins is critical for their association with membrane microdomains (8), while the presence of a 24:0 or C24:1 fatty acid chain in glycosphingolipids has been demonstrated to be necessary for their functional connection with Src family members in membrane microdomains (6,9). In contrast, GPI-anchored receptor clusters transiently recruit Src family kinases, leading to their temporary immobilization and consequent activation (10,11).…”